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Whose Gene Is It Anyway? The Effect of Preparation Purity on Neutrophil Transcriptome Studies.

Thomas HB, Moots RJ, Edwards SW, Wright HL - PLoS ONE (2015)

Bottom Line: The level of contamination was assessed by morphology and flow cytometry.RNA-seq analysis identified only 25 genes that were significantly differentially-expressed between Polymorphprep and negatively-selected neutrophils across all three treatment groups (untreated, GM-CSF, TNFα).The expression levels of 34 cytokines/chemokines both before and after GM-CSF or TNFα treatment were not significantly different between neutrophil isolation methods and therefore not affected by contributions from non-neutrophil cell types.

View Article: PubMed Central - PubMed

Affiliation: Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.

ABSTRACT
Protocols for the isolation of neutrophils from whole blood often result in neutrophil preparations containing low numbers (~5%) of contaminating leukocytes, and it is possible that these contaminating cells contribute to highly sensitive assays that measure neutrophil gene expression (e.g. qPCR). We investigated the contribution of contaminating leukocytes on the transcriptome profile of human neutrophils following stimulation with inflammatory cytokines (GM-CSF, TNFα), using RNA-Seq. Neutrophils were isolated using Polymorphprep or the StemCell untouched neutrophil isolation kit (negative selection of "highly pure" neutrophils). The level of contamination was assessed by morphology and flow cytometry. The major source of contamination in Polymorphprep neutrophil preparations was from eosinophils and was highly donor dependent. Contaminating cells were largely, but not completely, absent in neutrophil suspensions prepared using negative selection, but the overall yield of neutrophils was decreased by around 50%. RNA-seq analysis identified only 25 genes that were significantly differentially-expressed between Polymorphprep and negatively-selected neutrophils across all three treatment groups (untreated, GM-CSF, TNFα). The expression levels of 34 cytokines/chemokines both before and after GM-CSF or TNFα treatment were not significantly different between neutrophil isolation methods and therefore not affected by contributions from non-neutrophil cell types. This work demonstrates that low numbers (<5%) of contaminating leukocytes in neutrophil preparations contribute very little to the overall gene expression profile of cytokine-stimulated neutrophils, and that protocols for the isolation of highly pure neutrophils result in significantly lower yields of cells which may hinder investigations where large numbers of cells are required or where volumes of blood are limited.

No MeSH data available.


Neutrophil yield using density gradient isolation or negative selection.(A) Neutrophil yield (106/mL) from whole blood isolated by Polymorphprep and negative selection (Beads). Data represents n = 5 paired neutrophil isolations (**p<0.01). (B) Neutrophil yield (106/mL) from whole blood isolated by Ficoll-Paque (Ficoll) and negative selection (Beads). Data represent n = 5 experiments in which neutrophil enrichment from the Ficoll-Paque granulocyte pellet was carried out using negative selection (**p<0.01).
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pone.0138982.g002: Neutrophil yield using density gradient isolation or negative selection.(A) Neutrophil yield (106/mL) from whole blood isolated by Polymorphprep and negative selection (Beads). Data represents n = 5 paired neutrophil isolations (**p<0.01). (B) Neutrophil yield (106/mL) from whole blood isolated by Ficoll-Paque (Ficoll) and negative selection (Beads). Data represent n = 5 experiments in which neutrophil enrichment from the Ficoll-Paque granulocyte pellet was carried out using negative selection (**p<0.01).

Mentions: In addition to purity levels, different isolation methods can yield significantly different numbers of neutrophils[4,5]. To quantify differences between Polymorphprep and negative selection of neutrophils, samples of whole blood from healthy donors (n = 5) were divided into two aliquots, and subjected to both methods of neutrophil isolation. Final preparations of neutrophils were counted using a coulter counter and the number of neutrophils recovered per mL of whole blood was calculated. Mean numbers of neutrophils obtained after negative selection were only ~40% of those recovered after Polymorphprep isolation using blood from the same donor (Fig 2A, p<0.01). This suggests that a large proportion of whole blood neutrophils are somehow “lost”during negative selection. The first step of the negative selection protocol is an osmotic depletion of erythrocytes by aggregation and sedimentation under atmospheric pressure. This typically produces a leukocyte-rich plasma layer which is ~50% of the initial volume of whole blood, which is subsequently used in the negative selection isolation. In order to investigate whether this may account for the number of neutrophils “lost” in the negative selection protocol, neutrophils were first isolated from whole blood of healthy donors (n = 5) using Ficoll-Paque. The number of cells in the granulocyte pellet were quantified and found to be ~95% neutrophils (data not shown). Highly-pure neutrophils were then enriched from the granulocyte pellet using negative selection. We again observed that ~50% of neutrophils were “lost” during the negative selection protocol (Fig 2B, p<0.01) suggesting that a population of neutrophils are being retained by the antibody:bead complex used in the negative selection protocol.


Whose Gene Is It Anyway? The Effect of Preparation Purity on Neutrophil Transcriptome Studies.

Thomas HB, Moots RJ, Edwards SW, Wright HL - PLoS ONE (2015)

Neutrophil yield using density gradient isolation or negative selection.(A) Neutrophil yield (106/mL) from whole blood isolated by Polymorphprep and negative selection (Beads). Data represents n = 5 paired neutrophil isolations (**p<0.01). (B) Neutrophil yield (106/mL) from whole blood isolated by Ficoll-Paque (Ficoll) and negative selection (Beads). Data represent n = 5 experiments in which neutrophil enrichment from the Ficoll-Paque granulocyte pellet was carried out using negative selection (**p<0.01).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581699&req=5

pone.0138982.g002: Neutrophil yield using density gradient isolation or negative selection.(A) Neutrophil yield (106/mL) from whole blood isolated by Polymorphprep and negative selection (Beads). Data represents n = 5 paired neutrophil isolations (**p<0.01). (B) Neutrophil yield (106/mL) from whole blood isolated by Ficoll-Paque (Ficoll) and negative selection (Beads). Data represent n = 5 experiments in which neutrophil enrichment from the Ficoll-Paque granulocyte pellet was carried out using negative selection (**p<0.01).
Mentions: In addition to purity levels, different isolation methods can yield significantly different numbers of neutrophils[4,5]. To quantify differences between Polymorphprep and negative selection of neutrophils, samples of whole blood from healthy donors (n = 5) were divided into two aliquots, and subjected to both methods of neutrophil isolation. Final preparations of neutrophils were counted using a coulter counter and the number of neutrophils recovered per mL of whole blood was calculated. Mean numbers of neutrophils obtained after negative selection were only ~40% of those recovered after Polymorphprep isolation using blood from the same donor (Fig 2A, p<0.01). This suggests that a large proportion of whole blood neutrophils are somehow “lost”during negative selection. The first step of the negative selection protocol is an osmotic depletion of erythrocytes by aggregation and sedimentation under atmospheric pressure. This typically produces a leukocyte-rich plasma layer which is ~50% of the initial volume of whole blood, which is subsequently used in the negative selection isolation. In order to investigate whether this may account for the number of neutrophils “lost” in the negative selection protocol, neutrophils were first isolated from whole blood of healthy donors (n = 5) using Ficoll-Paque. The number of cells in the granulocyte pellet were quantified and found to be ~95% neutrophils (data not shown). Highly-pure neutrophils were then enriched from the granulocyte pellet using negative selection. We again observed that ~50% of neutrophils were “lost” during the negative selection protocol (Fig 2B, p<0.01) suggesting that a population of neutrophils are being retained by the antibody:bead complex used in the negative selection protocol.

Bottom Line: The level of contamination was assessed by morphology and flow cytometry.RNA-seq analysis identified only 25 genes that were significantly differentially-expressed between Polymorphprep and negatively-selected neutrophils across all three treatment groups (untreated, GM-CSF, TNFα).The expression levels of 34 cytokines/chemokines both before and after GM-CSF or TNFα treatment were not significantly different between neutrophil isolation methods and therefore not affected by contributions from non-neutrophil cell types.

View Article: PubMed Central - PubMed

Affiliation: Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.

ABSTRACT
Protocols for the isolation of neutrophils from whole blood often result in neutrophil preparations containing low numbers (~5%) of contaminating leukocytes, and it is possible that these contaminating cells contribute to highly sensitive assays that measure neutrophil gene expression (e.g. qPCR). We investigated the contribution of contaminating leukocytes on the transcriptome profile of human neutrophils following stimulation with inflammatory cytokines (GM-CSF, TNFα), using RNA-Seq. Neutrophils were isolated using Polymorphprep or the StemCell untouched neutrophil isolation kit (negative selection of "highly pure" neutrophils). The level of contamination was assessed by morphology and flow cytometry. The major source of contamination in Polymorphprep neutrophil preparations was from eosinophils and was highly donor dependent. Contaminating cells were largely, but not completely, absent in neutrophil suspensions prepared using negative selection, but the overall yield of neutrophils was decreased by around 50%. RNA-seq analysis identified only 25 genes that were significantly differentially-expressed between Polymorphprep and negatively-selected neutrophils across all three treatment groups (untreated, GM-CSF, TNFα). The expression levels of 34 cytokines/chemokines both before and after GM-CSF or TNFα treatment were not significantly different between neutrophil isolation methods and therefore not affected by contributions from non-neutrophil cell types. This work demonstrates that low numbers (<5%) of contaminating leukocytes in neutrophil preparations contribute very little to the overall gene expression profile of cytokine-stimulated neutrophils, and that protocols for the isolation of highly pure neutrophils result in significantly lower yields of cells which may hinder investigations where large numbers of cells are required or where volumes of blood are limited.

No MeSH data available.